The invention relates generally to detectable labels and compositions useful in assay methods for detecting soluble, suspended, or particulate substances or analytes such as proteins, carbohydrates, nucleic acids, bacteria, viruses, and eukaryotic cells and more specifically relates to compositions and methods that include luminescent (e.g., phosphorescent) labels.
A detectable phosphor label is typically a phosphor conjugated with capture molecules that are specific for analytes of interest. Detectable phosphor labels can be used in all assay applications where fluorochrome, enzyme, or isotope-labelled immuno-reagents are used. Various phosphor conjugates, their preparation, and use were previously described in, for example, U.S. Pat. No. 5,043,265 (Tanke et al.), the disclosure of which is incorporated herein by reference. Examples of assay applications are enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) techniques, and lateral flow assays for demonstrating and assaying an analyte in solution, immunological methods for the detection of macromolecules in filter blots, immunocytochemical methods for the study of morphologically intact tissues and cells, etc. As for the cytochemical applications, they usually target superficial antigens, as phosphor particles of 0.1-1.0 μm cannot easily penetrate cell membranes.
By using phosphors as detectable labels in an assay, several parameters can be studied and measured at the same time. Not only is it possible to generate three spectrally separate colours (blue, green, red) by means of infra red (IR), ultra violet (UV), or electron excitation, for example, but phosphor emission wavelengths and intensity aplitudes can be measured, and the number of analytes to be measured at the same time can become very large (multiplexing). Time-resolved luminescence assays are comparable in sensitivity to radioactivity assays. The immunocytochemical use of phosphor conjugates with capture molecules (analyte-specific phosphor conjugates) allows a much more sensitive detection of small quantities of macromolecules in cells. This may be of importance in both fundamental and diagnostic examination in various applications.
Examples of the properties of the phosphors, other than their high physico-chemical stability, are that they can be rendered visible by excitation with, for example, IR excitation, UV light, or with an electron beam, and that the luminescence of the phosphor-capture molecule conjugates, such as phosphor-antibody conjugates, does not decrease during excitation (no bleaching). In addition, the luminescence belongs to the relatively slow luminescence (phosphorescence). The luminescence of phosphors can be observed with microscope fluorimeters and flow cytometers. These can be modified for time-resolved luminescence assays in a relatively simple manner. The use of phosphor-capture molecule conjugates makes it possible to assay a plurality of analytes simultaneously, because the luminescence of phosphors is not only well separated spectrally (blue, green, red), but also exhibits measurable differences in decay times.
Prior multiplexing was generally performed by selective excitation and/or detecting the emission wavelength of the different phosphors. Simultaneous detection of multiple phosphors is possible, at least where the phosphors have the same excitation bands or different emission bands.
Currently there is a need for more rapid, ultrasensitive, and specific assays and methods that can image and detect multiple analytes in a sample in a single test assay readout. Because prior phosphor particles were not uniform in their morphology, size, and/or composition, it was not possible to detect analytes based on the unique optical lifetime signature of each type of phosphor in the conjugate being used as detectable label in an assay.